H2N2 cold-adapted (ca) vaccine: H2N2 viruses caused the 1957 influenza pandemic and circulated in humans until 1968 when they were replaced by H3N2 viruses. Although H2 viruses have not circulated in humans since 1968, this subtype is maintained in avian reservoirs worldwide. An H2 vaccine is a high priority in preparing for a pandemic because H2 viruses have a proven capability for causing disease in humans and persons born after 1968 lack H2-specific immunity. Because the currently licensed live attenuated influenza vaccine utilized in the United States bears the internal protein genes of the influenza A/AnnArbor/6/60 (H2N2) ca virus, the A/AnnArbor/6/60 (H2N2) ca virus was a logical first choice for evaluation as an H2 vaccine candidate. However, this virus with the H2 HA and N2 NA had not been evaluated in seronegative people. An IND was submitted for a Phase I study to evaluate safety, level of replication, infectivity and immunogenicity of A/Ann Arbor/6/60 ca (H2N2) virus. We evaluated the safety, infectivity, and immunogenicity of two doses of 7log TCID50 of this vaccine administered by nasal spray 4 weeks apart to normal healthy seronegative adults in an inpatient isolation unit. The participants were followed for 2 months after 1 dose of vaccine or for 4 weeks after the second dose. Twenty-one participants received a first dose of the vaccine, and 18 participants received a second dose 4 weeks later. No serious adverse events occurred during the trial. The most common adverse events after vaccination were headache and musculoskeletal pain. The vaccine was restricted in replication: 5 participants (24%) had virus detectable by culture or by rRT-PCR after the first dose, and 3 participants (17%) had virus detectable by culture or by rRT-PCR after the second dose. Antibody responses to the vaccine were also restricted: 24% of participants developed an antibody response as measured by either hemagglutination-inhibition assay (10%), or ELISA for H2 HA-specific serum IgG (24%) or IgA (16%) after either one or two doses. None of the participants had a neutralizing antibody response. Following the first dose, vaccine specific IgG secreting cells as measured by ELISPOT increased from a mean of 0.5 to 2.0/million PBMCs; vaccine specific IgA secreting cells increased from 0.1 to 0.5/million PBMCs. In conclusion, the live attenuated H2N2 1960 AA ca vaccine demonstrated a safety profile consistent with seasonal trivalent LAIV but was restricted in replication and minimally immunogenic in healthy seronegative adults. H2N3 cold-adapted (ca) vaccine: Based on promising preclinical data in mice and ferrets, an IND was submitted for a Phase I study to evaluate safety, level of replication, infectivity and immunogenicity of an H2N3 ca vaccine based on A/swine/MO/2006 (H2N3). Nineteen subjects received the first dose and 15 received two doses of the vaccine. The vaccine was well-tolerated and the analysis of the data is in progress. H7N7 cold-adapted (ca) vaccine: Based on promising preclinical data in mice and ferrets, an IND was submitted for a Phase I study to evaluate safety, level of replication, infectivity and immunogenicity of an H7N7 ca vaccine based on A/Netherlands/219/2003 (H7N7). Fifteen subjects received the first dose and 13 received two doses of the vaccine. The vaccine was well-tolerated and the analysis of the data is in progress. H7N3 cold-adapted (ca) vaccine: We had previously evaluated the safety, infectivity, and immunogenicity of two doses of log7.5 TCID50 of the vaccine administered by nasal spray 5 weeks apart to normal healthy seronegative adult volunteers in an inpatient isolation unit. The subjects were followed for 2 months after 1 dose of vaccine or for 4 weeks after the second dose. The live attenuated H7N3 vaccine was well-tolerated but was highly restricted in replication in healthy seronegative adults. Despite the restricted replication, the vaccine was immunogenic, with serum IgA being the most sensitive measure of immunogenicity. A study to evaluate the safety, level of replication, infectivity and immunogenicity of a single dose of the H7N3 ca vaccine was undertaken. Twenty subjects received a single dose of the vaccine. The vaccine was well-tolerated and the analysis of the data is in progress.